The present invention relates moreover to an apparatus for examining a sample, the apparatus having a light source for generating excitation light in pulses which occur in succession at an excitation pulse frequency, for illuminating a sample region with the excitation pulse, and having a detector for detecting the detection light emanating from the sample region.
To investigate the properties or the dynamic behavior of biological samples, many applications require that they be exposed to illuminating light in order to subsequently analyze the detection light emanating from the sample. Excitation light is often used to optically excite fluorescent dyes, for example, in order to subsequently investigate the properties of the fluorescent light emanating from the sample. Some examination methods call for using a pulsed light instead of a continuous light beam to illuminate the sample; for example for measuring the lifetime of an excitation state. It is namely possible, for example, to excite fluorescent dyes with short light pulses, in order to electronically measure the time delay of the emission light pulses. German Patent Application DE 10 2004 017 956 A1, for example, describes a microscope for analyzing the lifetime of excited states in a sample. The microscope includes at least one light source for generating excitation light and at least one detector for receiving detection light emanating from the sample. The microscope is characterized in that the light source includes a semiconductor laser which emits pulsed excitation light, an adjusting device being provided for adjusting the pulse repetition rate to the specific lifetime properties of the sample.
By analyzing the lifetime of the excited states of a sample labeled with one or more fluorescent dyes, important information can be obtained about the properties of the sample. In particular, information about a sample region being analyzed, such as information about the composition and surroundings thereof, can be obtained by using multiple fluorescent dyes with the aid of fluorescence lifetime imaging microscopy (FLIM). In cell biology, for example, the calcium concentration in a sample region can be indirectly inferred by measuring the lifetime of the fluorescent dyes.